A method and system for separating and determining the residual amount of paclobutrazol and uniconazole enantiomers in apples by ultra-high performance chromatography

Abstract

The present application relates to the technical field of pesticide residue detection, and particularly relates to a method and system for separating and determining the enantiomer residue amounts of paclobutrazol and uniconazole in apples by using ultra-high performance combined phase chromatography. The method comprises the following steps: extracting an apple sample by using acetonitrile, redissolving the extracted solution by using a methanol-dichloromethane mixed solution after concentration, purifying the extracted solution by using an NH2 solid phase extraction column, and then performing constant volume by using n-heptane after blowing dry; then, performing gradient elution by using an Acquity Trefoil CEL1 chiral chromatographic column under the conditions of 31 DEG C and 20.7 MPa, with carbon dioxide as a main mobile phase and 0.5% (v / v) ammonia water-methanol solution as a cosolvent, and then performing separation and detection on (+) paclobutrazol, (-) paclobutrazol, (+) uniconazole and (-) uniconazole by using a PDA detector, and then performing quantification by using an external standard method. The present application can realize good separation of four enantiomers in a short time, has the advantages of high sensitivity, good accuracy, good repeatability and less consumption of organic solvents, and is suitable for rapid detection and quality safety evaluation of chiral plant growth regulators in apples.

Description

Technical Field

[0001] This invention relates to the field of pesticide residue detection technology, and in particular to a method and system for separating and determining the enantiomeric residues of paclobutrazol and uniconazole in apples using ultra-high performance phase chromatography. Background Technology

[0002] Paclobutrazol and uniconazole are both triazole plant growth regulators, widely used in fruit trees, vegetables, and other cash crops for shoot control, flowering promotion, fruit retention, and plant architecture regulation. These compounds are characterized by low dosage, significant regulatory effects, and long-lasting effects in agricultural production, leading to their frequent use in apple and other fruit cultivation. However, because paclobutrazol and uniconazole may leave residues in fruits and their surface tissues after application, and enter consumer markets through harvesting, transportation, and distribution, their residue risks and detection control issues have received continuous attention from regulatory authorities and the food safety research field. The current national food safety standard GB 2763-2021 has stipulated residue limits for paclobutrazol in some foods, including the maximum residue limit for paclobutrazol in apples, indicating a clear practical need and standard basis for monitoring the residues of this type of plant growth regulator in apples.

[0003] Furthermore, paclobutrazol and uniconazole are not simply single-structure compounds, but rather chiral agrochemicals. Different enantiomers of chiral pesticides are mirror images of each other in spatial configuration. Although their physicochemical properties are very similar, they often exhibit significant differences in biological activity, plant regulatory effects, metabolic transformation behavior, environmental migration patterns, and ecotoxicity. In other words, even with the same total amount of racemic mixture, different proportions of enantiomers can lead to significant differences in actual efficacy, safety, and environmental risk assessment results. Therefore, evaluating chiral plant growth regulators solely based on total residues or conventional residue values ​​without configuration differentiation is insufficient to comprehensively and accurately reflect the true presence of highly active enantiomers and less active or even high-risk enantiomers in the target sample. In recent years, research on the enantiomeric selectivity behavior and detection methods of chiral pesticides has been increasing. Relevant review literature has clearly pointed out that qualitative and quantitative analysis of chiral pesticides should be conducted at the enantiomer level to support more refined efficacy evaluation and risk control.

[0004] For the detection of chiral pesticides, traditional analytical methods often employ gas chromatography, high-performance liquid chromatography, or liquid chromatography-mass spectrometry (LC-MS) to perform total analysis of the target compound or residue determination under achiral conditions. While these methods are widely used in routine pesticide residue detection, when the analytes are compounds with similar structures, polarities, and chiral configurations, traditional achiral chromatographic systems often struggle to achieve rapid, stable, and high-resolution separation between enantiomers. This is especially true in real food matrices like apples, where fructose, organic acids, pigments, waxes, phenols, and other endogenous components can easily co-enter the detection system with the analyte, leading to baseline fluctuations, poor peak shape, decreased resolution, or insufficient quantitative accuracy. The applicability of traditional methods is further limited when it is necessary to distinguish between paclobutrazol and uniconazole, and further differentiate their corresponding enantiomers.

[0005] In recent years, ultra-high performance convergence chromatography (UPC) has become increasingly important. 2 Supercritical fluid chromatography (SCLC), using supercritical or near-supercritical carbon dioxide as the main mobile phase, offers advantages such as high separation efficiency, fast analysis speed, low organic solvent consumption, and applicability to chiral separation, and has gradually become an important technical direction for chiral pesticide analysis. Existing technical literature (Abad-Gil L, et al. Enantioselective analysis of pesticides in food, biological and environmental matrices[J]) is relevant. Current Opinion in Food Science (2023.) systematically summarizes the enantiomeric analysis methods for chiral pesticides in food, biological, and environmental samples, pointing out that UPC 2 The use of chiral columns such as Trefoil CEL1 for enantiomeric separation of some chiral pesticides demonstrates the high potential of this technique for rapid separation of chiral compounds. This literature also indicates that the analysis of chiral pesticides has evolved from simply detecting them to achieving accurate separation and quantification at the enantiomeric level. The detection methods are highly dependent on sample pretreatment, purification methods, selection of chiral columns, co-solvent systems, column temperature, system back pressure, and detection modes.

[0006] However, while existing technologies provide methodological directions for the analysis of chiral pesticides, they still have significant shortcomings. First, current publications largely focus on describing specific types of chiral pesticides, specific matrices, or general chromatographic separation conditions, lacking specific methods for the simultaneous enantiomeric separation and residue determination of paclobutrazol and uniconazole, two triazole plant growth regulators, in apple matrices. Paclobutrazol and uniconazole have similar structures, UV absorption behaviors, and chromatographic retention characteristics; achieving stable separation of four enantiomeric peaks within the same analytical window is inherently more challenging than analyzing a single compound or a single enantiomeric component. Second, the composition of co-extracts varies considerably among different fruit matrices. Existing pretreatment and purification methods suitable for environmental samples, soil samples, or other food matrices may not be directly applicable to apple samples and may not yield the same good recoveries and precision. Furthermore, even given that UPC² is known to be suitable for chiral pesticide analysis, there is a lack of specific solutions in the existing technology for establishing a synergistic matching relationship between chiral column type, cosolvent type, system back pressure, column temperature, gradient elution program and volume adjustment reagent to simultaneously consider the resolution, peak shape, detection sensitivity and actual matrix adaptability of the four enantiomers.

[0007] Furthermore, from the perspective of food safety supervision and practical testing applications, the current standard system mainly sets limits for substances such as paclobutrazol based on total residue levels, while the limits and testing rules for specific enantiomers are insufficient. In other words, the regulatory need objectively exists, but the testing technology has not yet been fully refined to the routine application at the enantiomer level. For fruit categories such as apples, which have large import and export volumes and a wide consumer base, if analytical methods that do not distinguish between enantiomers are still used, it will be difficult to further identify which configuration component is dominant in the sample, and it will also be detrimental to subsequent differentiated assessments of highly active enantiomers, less active enantiomers, and potentially high-risk enantiomers. Therefore, establishing a method for apple samples that can simultaneously achieve rapid separation and accurate determination of the four enantiomers of paclobutrazol and uniconazole, with low limits of quantitation and good recovery rates, has become an urgent technical problem to be solved in the field of chiral plant growth regulator residue analysis.

[0008] In summary, while existing technologies have recognized the importance of enantiomeric analysis of chiral pesticides and have disclosed UPCs... 2 While technologies like paclobutrazol and uniconazole have shown promise in chiral separation, a dedicated analytical method is still lacking for the simultaneous detection of paclobutrazol and uniconazole enantiomers in apples, which balances sample pretreatment suitability, purification effectiveness, chiral separation efficiency, and quantitative accuracy. Therefore, it is necessary to develop a novel ultra-high performance phase chromatography (UHPLC) method for separating and determining the enantiomers of paclobutrazol and uniconazole in apples to meet the practical needs of refined detection and food safety evaluation of these chiral plant growth regulator enantiomers in apple samples. Summary of the Invention

[0009] The purpose of this invention is to address the problems in existing technologies for detecting paclobutrazol and uniconazole residues in apple samples, which can only perform total analysis, are difficult to achieve simultaneous enantiomeric separation and separate quantification, and suffer from insufficient separation, low sensitivity, and poor quantitative accuracy in complex fruit matrices. This invention provides a method and system for separating and determining the enantiomeric residues of paclobutrazol and uniconazole in apples using ultra-high performance phase chromatography (UHPLC). This method achieves rapid, stable separation and accurate determination of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole in apples through synergistic optimization of sample extraction and purification conditions, chiral chromatographic columns, mobile phase systems, co-solvent composition, system back pressure, column temperature, and gradient elution programs. This provides technical support for the refined residue analysis, quality and safety evaluation, and regulatory detection of chiral plant growth regulators in fruits.

[0010] Firstly, in order to achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for separating and determining the enantiomers of paclobutrazol and uniconazole in apples using ultra-high performance phase chromatography, comprising the following steps: S1. Sample extraction: Weigh 5g of the sample into a 50mL centrifuge tube, add 20mL of acetonitrile and 3g of sodium chloride, vortex to mix, shake to extract for 20min, centrifuge at 8500r / min for 5min, and take the supernatant into another 50mL centrifuge tube; add 20mL of acetonitrile to the lower residue, repeat the extraction once, and combine the supernatants from the two extractions. S2, Resolution and Purification: Add a mixed solution of methanol and dichloromethane in a volume ratio of 10:90 to the concentrated residue obtained in step S1 for resolution. Load the resolution solution onto an amino solid-phase extraction column activated by the mixed solution of methanol and dichloromethane in a volume ratio of 10:90. Collect the eluent and wash with the mixed solution of methanol and dichloromethane in a volume ratio of 10:90. Combine the eluent and wash and dry. S3, Volume adjustment: Dissolve the residue after drying in step S2 in n-heptane and adjust the volume, then filter to obtain the test solution; S4. Chiral separation and detection: The test solution was injected into an ultra-high performance phase chromatography system, and separation was performed using an AcquityTrefoil CEL1 chiral column. Carbon dioxide was used as mobile phase A, and 0.5% (v / v) ammonia-methanol solution was used as mobile phase B. Gradient elution was performed under the following conditions: column temperature 31℃, system back pressure 20.7MPa, flow rate 1.5mL / min, detection wavelength 220nm, and injection volume 5.0μL. The gradient elution program is as follows: 0-1.5 min, B is 3%; 1.5-2.0 min, B increases from 3% to 10%; 2.0-2.5 min, B is 10%; 2.5-2.6 min, B increases from 10% to 15%; 2.6-3.0 min, B is 15%; 3.1-3.5 min, B returns to 3%. S5. Qualitative and quantitative analysis: Based on the retention times of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole and (-)-uniconazole standards, the four enantiomers in the test solution were qualitatively analyzed, and their residual amounts were calculated using the external standard method.

[0011] Preferably, the amino solid-phase extraction column in step S2 is an NH2 solid-phase extraction column with a packing amount of 500 mg and a column volume of 3 mL.

[0012] Preferably, in step S2, the combined effluent and eluent are dried with nitrogen in a 40°C water bath.

[0013] Preferably, organic phase membrane filtration is used for filtration in step S3.

[0014] Preferably, the Acquity Trefoil CEL1 chiral column described in step S4 has dimensions of 150 mm × 3.0 mm and a diameter of 2.5 μm.

[0015] Preferably, the external standard method in step S5 uses a mixed standard working solution of four enantiomers to establish a standard curve, and each enantiomer is subjected to linear regression in the concentration range of 0.5~20.0 mg / L.

[0016] Preferably, the limit of quantitation for (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole and (-)-uniconazole is 0.1 mg / kg.

[0017] As a preferred option, the racemic standard stock solution is prepared as follows: Accurately weigh 0.01 g of paclobutrazol and uniconazole racemic standard, accurate to 0.1 mg, dissolve in isopropanol and dilute to 10 mL to prepare a 1.0 g / L racemic standard stock solution; Preparation of standard intermediate solutions of paclobutrazol and uniconazole racemic mixtures: Accurately pipette a certain amount of racemic standard stock solution and dilute with isopropanol to a standard intermediate solution of 20.0 mg / L; Preparation of enantiomeric standard stock solution: Accurately weigh 0.01 g of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole standards, accurate to 0.1 mg, dissolve in isopropanol and dilute to 10 mL to prepare an enantiomeric standard stock solution of 1.0 g / L. Preparation of mixed standard working solutions of four paclobutrazol and uniconazole enantiomers: Accurately pipette a certain amount of the (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole enantiomer standard stock solutions, and dilute them stepwise with isopropanol to 0.50, 1.00, 2.00, 4.00, 10.0, and 20.0 mg / L respectively to prepare mixed standard working solutions.

[0018] Preferably, the method showed recoveries of 80.0%–107% for the four enantiomers in apple samples at spiked levels of 0.1 mg / kg, 0.2 mg / kg, and 1.0 mg / kg, with relative standard deviations of 2.8%–8.4%.

[0019] Secondly, the present invention also provides an ultra-high performance chromatographic detection system for implementing the method, comprising: Sample extraction unit, purification unit, volume adjustment unit, and ultra-high performance phase chromatography detection unit; The sample extraction unit is used to perform acetonitrile double extraction and salting-out separation on apple samples. The purification unit is used to purify the concentrated and reconstituted extract with NH2 solid-phase extraction. The volume-degrading unit is used to dilute the purified sample to a final volume using n-heptane; The ultra-high performance phase chromatography detection unit includes a carbon dioxide supply module, a cosolvent supply module, a back pressure control module, a column temperature control module, an Acquity Trefoil CEL1 chiral chromatographic column, and a PDA detector, used to achieve the separation, detection, and quantitative analysis of four enantiomers of paclobutrazol and uniconazole in apples under the chromatographic conditions defined in claim 1.

[0020] This invention provides an ultra-high performance co-phase chromatography method and system for the separation and determination of paclobutrazol and uniconazole in apple samples. Addressing the challenge of detecting these structurally similar, chiral enantiomers that are easily interfered with by the fruit matrix, this method synergistically combines acetonitrile double extraction, NH2 solid-phase extraction purification, n-heptane volume adjustment, Acquity Trefoil CEL1 chiral column separation, a supercritical carbon dioxide / 0.5% (v / v) ammonia-methanol co-solubilizing system, a column temperature of 31℃, a system back pressure of 20.7 MPa, and a specific gradient elution program. This enables good baseline separation of the four enantiomers (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole within a short analysis time, with sharp peak shapes, high resolution, and good repeatability. Furthermore, this invention effectively reduces the impact of co-extraction of impurities in the apple matrix on the detection of the target analyte, resulting in high sensitivity and good quantitative accuracy within the range of 0.5–20.0 MPa. The invention exhibits good linearity within the mg / L range, with a high correlation coefficient and a limit of quantitation of 0.1 mg / kg. It achieves high recoveries and low relative standard deviations at low, medium, and high spiking levels, thus meeting the practical detection requirements for enantiomeric residue analysis of paclobutrazol and uniconazole in apples. Furthermore, compared to traditional liquid chromatography methods, this invention offers advantages such as faster analysis speed, lower organic solvent consumption, lower detection costs, and suitability for rapid screening and regulatory applications of batch samples. It can provide reliable technical support for the refined evaluation, risk identification, quality control, and import / export food safety supervision of chiral plant growth regulator residues in fruits. Attached Figure Description

[0021] Figure 1 The following are the molecular structural formulas of paclobutrazol and uniconazole. Wherein: (A) paclobutrazol; (B) uniconazole.

[0022] Figure 2 The effect of different chromatographic columns on the separation of enantiomers of four paclobutrazols and uniconazoles was investigated.

[0023] Figure 3 The effects of different separation conditions on the separation of four paclobutrazol and uniconazole enantiomers were investigated. Among them: (A) isocratic separation condition 1; (B) gradient separation condition 2; (C) gradient separation condition 3; (D) gradient separation condition 4; (E) gradient separation condition 5.

[0024] Figure 4 The effect of different co-solvents on the enantiomeric separation of four paclobutrazols and uniconazoles was investigated. The co-solvents included: (A) methanol; (B) 0.5% (v / v) formic acid in methanol solution; and (C) 0.5% (v / v) ammonia in methanol solution.

[0025] Figure 5 The effect of different system back pressures on the enantiomeric separation of four paclobutrazol and uniconazole.

[0026] Figure 6 The effect of different column temperatures on the separation of enantiomers of four paclobutrazols and uniconazoles was investigated.

[0027] Figure 7 The effect of different dilution reagents on the enantiomeric separation of four paclobutrazols and uniconazoles was investigated. The reagents used were: (A) methanol, (B) acetonitrile, (C) n-heptane, (D) isopropanol, and (E) anhydrous ethanol.

[0028] Figure 8 The effect of different purification methods on the purification efficiency of paclobutrazol and uniconazole enantiomers was investigated. The chromatographic peaks were: peak 1: (+)-paclobutrazol; peak 2: (-)-paclobutrazol; peak 3: (+)-uniconazole; peak 4: (-)-uniconazole.

[0029] Figure 9 Chromatograms of the standard solution (A), blank apple sample (B), and added recovery (C) are shown. Peak 1: (+)-Paclobutrazol; Peak 2: (-)-Paclobutrazol; Peak 3: (+)-Uniconazole; Peak 4: (-)-Uniconazole.

[0030] Figure 10 The resolution of paclobutrazol and uniconazole racemic mixtures is shown below. The solutions are: (A) standard solutions of paclobutrazol and uniconazole racemic mixtures; (B) (+)-paclobutrazol standard solution; (C) (-)-paclobutrazol standard solution; (D) (+)-uniconazole standard solution; (E) (-)-uniconazole standard solution. Chromatographic peak 1: (+)-paclobutrazol; chromatographic peak 2: (-)-paclobutrazol; chromatographic peak 3: (+)-uniconazole; chromatographic peak 4: (-)-uniconazole.

[0031] Figure 11 The chromatogram shows the positive apple sample. Peak 1: (+)-Paclobutrazol; Peak 2: (-)-Paclobutrazol; Peak 3: (+)-Uniconazole; Peak 4: (-)-Uniconazole. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0033] It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Any equivalent substitutions or conventional adjustments made to the types and amounts of raw materials, operating sequences, instrument models, detection parameters, and data processing methods based on the concept of the present invention, without departing from the spirit and essence of the present invention, shall fall within the scope of protection of the present invention.

[0034] This invention provides a method and system for separating and determining the enantiomers of paclobutrazol and uniconazole in apples using ultra-high performance combined phase chromatography (UHPLC). The method mainly includes sample extraction, sample purification, volume adjustment, chiral separation and detection by UHPLC, and quantification using external standard method. Through extensive experimental screening, this invention establishes an analytical method suitable for the simultaneous separation and accurate quantification of four enantiomers (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole in apple samples by synergistically combining acetonitrile extraction, amino solid-phase extraction purification, n-heptane volume adjustment, separation using an Acquity Trefoil CEL1 chiral column, a supercritical carbon dioxide / 0.5% (v / v) ammonia-methanol dissolution system, a column temperature of 31℃, a back pressure of 20.7 MPa, and a specific gradient elution program.

[0035] In the following examples, unless otherwise specified, the instruments, equipment, reagent purity, standard sources, and pretreatment equipment used are all products conventionally available in the art, as detailed below: I. Reagents, Standards and Instruments 1.1 Standard Products The purity of the racemic paclobutrazol standard is not less than 95.2%; the purity of the racemic uniconazole standard is not less than 99.5%; and the purity of the enantiomeric standards of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole are all not less than 95.0%.

[0036] 1.2 Organic reagents Acetonitrile, methanol, isopropanol, n-heptane, anhydrous ethanol, and dichloromethane were all of chromatographic purity. Sodium chloride was of analytical grade. High-purity carbon dioxide, with a purity of not less than 99.999%.

[0037] 1.3 Cleanroom Materials NH2 solid-phase extraction column, 500 mg / 3 mL; The C used in the comparative example 18 Both the solid-phase extraction column and the WCX solid-phase extraction column were used, with a concentration of 500 mg / 3 mL.

[0038] 1.4 Main Instruments Ultra-high performance phase chromatograph with diode array detector; electronic balance; rotary evaporator; nitrogen blower; vortex mixer; horizontal shaker; centrifuge; microporous membrane filtration device.

[0039] II. Preparation of Standard Solutions 2.1 Preparation of racemic standard stock solution Accurately weigh 0.0100 g each of paclobutrazol racemic standard and uniconazole racemic standard, accurate to 0.1 mg, and place them in 10 mL volumetric flasks respectively. Dissolve them in isopropanol and dilute to the mark to obtain paclobutrazol racemic standard stock solution and uniconazole racemic standard stock solution with a concentration of 1.0 g / L.

[0040] 2.2 Preparation of enantiomeric standard stock solutions Accurately weigh 0.0100 g each of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole and (-)-uniconazole standards, dissolve them in isopropanol and dilute to 10 mL to obtain four enantiomeric standard stock solutions with a concentration of 1.0 g / L.

[0041] 2.3 Preparation of mixed standard working solution Take appropriate amounts of the four enantiomer standard stock solutions and dilute them stepwise with isopropanol to obtain mixed standard working solutions with concentrations of 0.50 mg / L, 1.00 mg / L, 2.00 mg / L, 4.00 mg / L, 10.0 mg / L and 20.0 mg / L, respectively, for plotting standard curves.

[0042] III. General Operating Steps of the Method of the Invention 3.1 Step S1: Apple Sample Extraction Weigh 5.00 g (accurate to 0.01 g) of homogenized apple sample into a 50 mL centrifuge tube, add 20 mL of acetonitrile, then add 3 g of sodium chloride. Vortex to mix, then extract horizontally for 20 min. After extraction, centrifuge at 8500 rpm for 5 min and collect the supernatant in another centrifuge tube. Add 20 mL of acetonitrile to the residue again, and repeat the above extraction and centrifugation steps once more. Combine the supernatants from both extractions.

[0043] 3.2 Step S2: Concentration, Reconstitution and Purification The combined extracts were concentrated to near dryness using a rotary evaporator. 3 mL of a methanol:dichloromethane (10:90 v / v) mixture was added to the concentrated residue for redissolution. The resulting redissol was transferred to an NH2 solid-phase extraction column pre-activated with a methanol:dichloromethane (10:90 v / v) mixture, and the eluent was collected directly. The column was then eluted with the same mixture, and the eluent was combined with the eluent. The combined solution was dried under nitrogen in a 40°C water bath.

[0044] 3.3 Step S3: Volume Adjustment Add 1.0 mL of n-heptane to the dried residue to dissolve and make up to volume. After mixing, filter through an organic phase filter membrane to obtain the test solution.

[0045] 3.4 Step S4: Chiral separation and detection by ultra-high performance phase chromatography An Acquity Trefoil CEL1 chiral column with dimensions of 150 mm × 3.0 mm and a diameter of 2.5 μm was used.

[0046] Mobile phase A is carbon dioxide, and mobile phase B is a 0.5% (v / v) ammonia-methanol solution.

[0047] The detection wavelength was 220 nm; the system back pressure was 20.7 MPa; the column temperature was 31 °C; the flow rate was 1.5 mL / min; and the injection volume was 5.0 μL. The gradient elution program was as follows: A is CO2, and B is a 0.5% (v / v) ammonia-methanol solution. Gradient separation conditions: 0~1.5 min (3%B), 1.5~2 min (3%~10%B), 2~2.5 min (10%B), 2.5~2.6 min (10%~15%B), 2.6~3 min (15%B), 3.1~3.5 min (3%B); 3.5 Step S5: Qualitative and Quantitative Analysis The retention times of enantiomer standards of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole and (-)-uniconazole were used as the qualitative basis. Standard curves were plotted with peak area against mass concentration. The residual amounts of the four enantiomers in apple samples were calculated by external standard method.

[0048] IV. Results and Discussion 4.1 Selection of detection wavelength After scanning with a PDA detector, the UV spectra of four paclobutrazol and uniconazole enantiomer standard solutions were extracted from the chromatograms. The results showed that paclobutrazol exhibited significant absorption in the 220–230 nm UV wavelength range, while uniconazole showed significant absorption in the 230–254 nm UV wavelength range. This study selected three UV wavelengths (210, 220, and 230 nm) for simultaneous chromatographic analysis of paclobutrazol and uniconazole. It was found that both plant growth regulators showed significant absorption at 220 nm, indicating suitability for simultaneous detection.

[0049] 4.2 Column Optimization This experiment selected four chiral separation columns—CHIRALPAK AD-3, IC, and OJ-H from Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd.—and Waters Acquity Trefoil CEL1 to investigate their separation efficiency for four paclobutrazol and uniconazole enantiomers. The results showed that when using the AD-3 and IC chiral columns, only three chromatographic peaks appeared in the chromatograms, and some compounds overlapped (see...). Figure 2 Compared to the OJ-H column, the CEL1 chiral column produced sharper peaks and earlier elution times for the four enantiomers. Further optimization of the system back pressure and column temperature could lead to even higher resolution. Therefore, the CEL1 chiral column was chosen for this experiment to separate the four paclobutrazol and uniconazole enantiomers.

[0050] 4.3 Selection of Chromatographic Separation Program To obtain the optimal chromatographic separation program, this application investigated the effects of different chromatographic separation programs on the separation of enantiomers of four paclobutrazols and uniconazole. The gradient elution program was as follows: A was CO2, and B was 0.5% (v / v) ammonia-methanol solution. Isocratic separation condition 1: 0~7 min (10% B); Gradient separation condition 2: 0~1.5 min (3%B), 1.5~2 min (3%~10%B), 2~2.5 min (10%B), 2.5~2.6 min (10%~15%B), 2.6~3 min (15%B), 3.1~3.5 min (3%B); Gradient separation condition 3: 0~1 min (5%B), 1~1.1 min (5%~10%B), 1.1~1.8 min (10%B), 1.8~1.9 min (10%~15%B), 1.9~2.5 min (15%B), 2.5~2.6 min (15%~5%B), 2.6~3 min (5%B); Gradient separation condition 4: 0~1.5 min (3%B), 1.5~1.6 min (3%~10%B), 1.6~2 min (10%B), 2~2.1 min (10%~15%B), 2.1~2.5 min (15%B), 2.5~2.6 min (15%~3%B), 3.1~3.5 min (3%B); Gradient separation condition 5: 0~1.5 min (3%B), 1.5~1.8 min (3%~10%B), 1.8~2 min (10%B), 2~2.1 min (10%~15%B), 2.1~2.5 min (15%B), 2.5~2.6 min (15%~3%B), 3.1~3.5 min (3%B).

[0051] The results showed that when isocratic separation condition 1 was used, the peaks of the four chromatographic peaks exhibited poor symmetry and were excessively broadened, lacking sharpness. Compared to other gradient separation conditions, gradient separation condition 2 resulted in better separation of the four chromatographic peaks and better peak symmetry (see...). Figure 3 B). Therefore, this experiment selects gradient separation condition 2.

[0052] 4.4 Optimization of Cosolvents Ultra-high performance phase chromatography (UHPLC) consumes less organic solvent and uses supercritical CO2 as the main mobile phase. A small amount of organic solvent is typically used as a co-solvent to enhance the elution capacity and selectivity of the target product. This experiment investigated the effects of different co-solvents, including methanol, 0.5% (v / v) formic acid in methanol solution, and 0.5% (v / v) ammonia in methanol solution, on the separation of four paclobutrazol and uniconazole enantiomers. The results showed that when 0.5% (v / v) formic acid in methanol solution was used as the co-solvent, the chromatographic baseline was uneven and the peaks were very small. Compared to methanol, when 0.5% (v / v) ammonia in methanol solution was used as the co-solvent, the chromatographic peak separation of the four paclobutrazol and uniconazole enantiomers was better (see...). Figure 4 Therefore, our laboratory selected a 0.5% (v / v) ammonia-methanol solution as a co-solvent.

[0053] 4.5 Optimization of System Back Pressure UPC 2 Supercritical CO2 was used as the mobile phase. Adjusting the system back pressure and temperature effectively altered the density of CO2, thereby changing its solubility, elution capacity, and selectivity. CO2 only enters the supercritical state when its temperature exceeds 31℃ and its pressure exceeds 7.38 MPa. This experiment investigated the effect of system back pressure in the range of 10.3–17.2 MPa on the separation of enantiomers of four paclobutrazols and uniconazole. The results showed that as the system back pressure increased, the retention time of the analytes decreased, while the peak shapes remained relatively similar (see...). Figure 5 In comparison, using 20.7 MPa as the system back pressure resulted in better chromatographic peak resolution. Therefore, 20.7 MPa was chosen as the system back pressure in this experiment.

[0054] 4.6 Column Temperature Optimization Considering that the maximum recommended operating temperature of the Acquity Trefoil CEL1 chiral column is 40℃, and CO2 only enters the supercritical CO2 state when the temperature exceeds 31℃ and the pressure exceeds 7.38 MPa, this experiment investigated the effect of column temperature in the range of 31~40℃ on the separation of four paclobutrazol and uniconazole enantiomers. Under the three column temperature conditions, the chromatographic peak resolution of the four paclobutrazol and uniconazole enantiomers was good, and good baseline separation was achieved within 3.4 min, with little difference in peak shape. Figure 6 In comparison, the chromatographic peak resolution of the four paclobutrazol and uniconazole enantiomers was better when the column temperature was 31℃. Therefore, 31℃ was selected as the column temperature for the experiment.

[0055] 4.7 Optimization of Volume Adjustment Reagent Five dilution reagents—methanol, acetonitrile, n-heptane, isopropanol, and anhydrous ethanol—were used to separate the enantiomers of 50 mg / L paclobutrazol and uniconazole. The results are as follows: Figure 7 As shown, compared with the other four dilution reagents, when n-heptane was used as the dilution reagent, the chromatographic peaks of the four paclobutrazol and uniconazole enantiomers were sharp and the separation was good. Therefore, n-heptane was used as the dilution reagent in the experiment.

[0056] 4.8 Investigation of different purification methods The experiment compared C 18 The purification effects of three purification methods—C column, NH2 column, and WCX column—on apple sample extract solutions were investigated. Four paclobutrazol and uniconazole enantiomer standard solutions were added to apple samples that did not contain paclobutrazol or uniconazole. The samples were extracted twice with acetonitrile by shaking. After the extracts were concentrated to dryness and reconstituted, they were treated using the three different purification methods. The results showed that compared to C… 18 When using NH2 column purification, the recovery rates of the four paclobutrazol and uniconazole enantiomers were higher in both NH2 and WCX columns (see [link to NH2 column purification process]). Figure 8 Therefore, the experiment ultimately selected the NH2 solid-phase extraction column as the purification column.

[0057] 4.9 Methodological Examination 4.9.1 Linear range and limit of quantitation A series of mixed standard solutions of paclobutrazol and uniconazole enantiomers were determined under the chromatographic conditions described above. A standard curve was plotted with the peak area (Y) of the standards on the ordinate and the corresponding mass concentration (X) on the abscissa, and the regression equation and correlation coefficient were obtained. The results showed that the four enantiomers exhibited good linearity in the concentration range of 0.5–20.0 mg / L, with a correlation coefficient greater than 0.9991. By adding the standards to apple blank samples containing neither paclobutrazol nor uniconazole, and determining the enantiomers according to this method, the limits of quantitation (LOQ) were calculated with a signal-to-noise ratio (S / N) = 10. The LOQs for (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole were all 0.1 mg / kg (see Table 1).

[0058] Table 1. Linear range, linear equation, correlation coefficient, and limit of quantitation for each compound.

[0059] 4.9.2 Recovery rate and precision Four enantiomer standard solutions of paclobutrazol and uniconazole at different concentrations were added to apple samples that did not contain paclobutrazol and uniconazole. Spiking recovery and precision tests were performed. The relevant chromatograms are shown below. Figure 9 The results are shown in Table 2. The results indicate that the recoveries of the four paclobutrazol and uniconazole enantiomers ranged from 80.0% to 107%, with relative standard deviations (RSDs) of 2.8% to 8.4%, which meet the recovery requirements of SANTE / 11312 / 2021 and can satisfy the determination of paclobutrazol and uniconazole enantiomers in apple samples.

[0060] Table 2. Spike recoveries and relative standard deviations of paclobutrazol and uniconazole enantiomers in apple samples (n=6)

[0061] 4.10 Application of the Method 4.10.1 Resolution of racemic mixtures The method established in this application was used to separate and determine the purchased racemic standards of paclobutrazol and uniconazole. Figure 10 As shown in Figure A, the separation of the four paclobutrazol and uniconazole enantiomers was good, achieving effective separation within 3.4 min, with resolutions of R = 1.6, 3.1, and 2.4, respectively, meeting the requirement of complete separation with R ≥ 1.5. According to the retention time order of the chromatographic peaks, they were: (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole. Figure 10B, 10C, 10D, 10E). Based on the standard curves plotted above, the enantiomers of four paclobutrazol and uniconazole in the 20.0 mg / L intermediate standard solution of paclobutrazol and uniconazole racemic mixtures in Section 2.1 were calculated using the external standard quantification method. The contents of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole were 9.89 mg / L, 10.3 mg / L, 10.2 mg / L, and 9.78 mg / L, respectively.

[0062] 4.10.2 Testing of actual samples To examine the effectiveness and practicality of this method, the established method was used to determine the contents of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole in 20 commercially available apple samples. The results showed that paclobutrazol and uniconazole enantiomers were not detected in 19 apple samples, while (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole were detected in 1 apple sample, with detected levels of 0.205 mg / kg, 0.210 mg / kg, 0.204 mg / kg, and 0.196 mg / kg, respectively (see [link to study]. Figure 11 ).

[0063] 4. Conclusion This application is the first to employ ultra-high performance phase chromatography (UHPLC) to simultaneously separate four enantiomers of paclobutrazol and uniconazole, and to determine the residual amounts of paclobutrazol and uniconazole enantiomers in apples. The method optimized instrumental separation conditions, including detection wavelength, chromatographic column, chromatographic separation program, co-solvent, system back pressure, and column temperature. A suitable purification column for pretreatment purification was selected. The established method was applied to the detection of actual samples. Results showed good linearity for paclobutrazol and uniconazole enantiomers in the concentration range of 0.5–20.0 mg / L, with linear correlation coefficients greater than 0.9991 and a limit of quantitation of 0.1 mg / kg. Spiking experiments were conducted at three concentration levels: high (1.0 mg / kg), medium (0.2 mg / kg), and low (0.1 mg / kg). The recoveries of paclobutrazol and uniconazole enantiomers ranged from 80.0% to 107%, with relative standard deviations ranging from 2.8% to 8.4%. This method exhibits high sensitivity and good stability, meeting the requirements for detecting chiral enantiomer residues of paclobutrazol and uniconazole in apples. It provides a reference for in-depth analysis of effective and low-toxicity enantiomers and ineffective and highly toxic pesticide enantiomer residues in dairy products, and is of significant importance for drug quality control and efficacy evaluation.

[0064] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined in this application may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown in this application, but is to be accorded the widest scope consistent with the principles and novelty disclosed in this application.

Claims

1. A method for separating and determining the enantiomeric residues of paclobutrazol and uniconazole in apples using ultra-high performance phase chromatography, characterized in that, Includes the following steps: S1. Sample extraction: Weigh 5g of the sample into a 50mL centrifuge tube, add 20mL of acetonitrile and 3g of sodium chloride, vortex to mix, shake to extract for 20min, centrifuge at 8500r / min for 5min, and take the supernatant into another 50mL centrifuge tube. Add 20 mL of acetonitrile to the lower layer residue, repeat the extraction once, and combine the two supernatants; S2, Resolution and Purification: Add a mixed solution of methanol and dichloromethane in a volume ratio of 10:90 to the concentrated residue obtained in step S1 for resolution. Load the resolution solution onto an amino solid-phase extraction column activated by the mixed solution of methanol and dichloromethane in a volume ratio of 10:

90. Collect the eluent and wash with the mixed solution of methanol and dichloromethane in a volume ratio of 10:

90. Combine the eluent and wash and dry. S3, Volume adjustment: Dissolve the residue after drying in step S2 in n-heptane and adjust the volume, then filter to obtain the test solution; S4. Chiral separation and detection: The test solution was injected into an ultra-high performance phase chromatography system, and separation was performed using an Acquity TrefoilCEL1 chiral column. Carbon dioxide was used as mobile phase A, and 0.5% (v / v) ammonia-methanol solution was used as mobile phase B. Gradient elution was performed under the following conditions: column temperature 31℃, system back pressure 20.7MPa, flow rate 1.5mL / min, detection wavelength 220nm, and injection volume 5.0μL. The gradient elution program is as follows: 0-1.5 min, B is 3%; 1.5-2.0 min, B increases from 3% to 10%; 2.0-2.5 min, B is 10%; 2.5-2.6 min, B increases from 10% to 15%; 2.6-3.0 min, B is 15%; 3.1-3.5 min, B returns to 3%. S5. Qualitative and quantitative analysis: Based on the retention times of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole and (-)-uniconazole standards, the four enantiomers in the test solution were qualitatively analyzed, and their residual amounts were calculated using the external standard method.

2. The method according to claim 1, characterized in that, The amino solid-phase extraction column mentioned in step S2 is an NH2 solid-phase extraction column with a packing amount of 500 mg and a column volume of 3 mL.

3. The method according to claim 1, characterized in that, In step S2, the combined effluent and eluent are dried with nitrogen under a 40°C water bath.

4. The method according to claim 1, characterized in that, In step S3, filtration is performed using an organic phase membrane filter.

5. The method according to claim 1, characterized in that, The Acquity Trefoil CEL1 chiral column mentioned in step S4 has a specification of 150mm × 3.0mm and 2.5μm.

6. The method according to claim 1, characterized in that, In step S5, the external standard method uses a mixed standard working solution of four enantiomers to establish a standard curve, and each enantiomer is subjected to linear regression in the concentration range of 0.5~20.0 mg / L.

7. The method according to claim 1, characterized in that, The limits of quantification for (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole are all 0.1 mg / kg.

8. The method according to claim 1, characterized in that, The racemic standard stock solution was prepared as follows: Accurately weigh 0.01 g of paclobutrazol and uniconazole racemic standard, accurate to 0.1 mg, dissolve in isopropanol and dilute to 10 mL to prepare a 1.0 g / L racemic standard stock solution; Preparation of standard intermediate solutions of paclobutrazol and uniconazole racemic mixtures: Accurately pipette a certain amount of racemic standard stock solution and dilute with isopropanol to a standard intermediate solution of 20.0 mg / L; Preparation of enantiomeric standard stock solution: Accurately weigh 0.01 g of (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole standards, respectively, to the nearest 0.1 mg, dissolve them in isopropanol and bring the volume to 10 mL to prepare an enantiomeric standard stock solution of 1.0 g / L. Preparation of mixed standard working solutions of four paclobutrazol and uniconazole enantiomers: Accurately pipette a certain amount of the (+)-paclobutrazol, (-)-paclobutrazol, (+)-uniconazole, and (-)-uniconazole enantiomer standard stock solutions, and dilute them stepwise with isopropanol to 0.50, 1.00, 2.00, 4.00, 10.0, and 20.0 mg / L respectively to prepare mixed standard working solutions.

9. The method according to claim 1, characterized in that, The method achieved recoveries of 80.0%–107% for the four enantiomers in apple samples at spiked levels of 0.1 mg / kg, 0.2 mg / kg, and 1.0 mg / kg, with relative standard deviations of 2.8%–8.4%.

10. An ultra-high performance phase-coherence chromatography detection system for implementing the method according to any one of claims 1-9, characterized in that, include: Sample extraction unit, purification unit, volume adjustment unit, and ultra-high performance phase chromatography detection unit; The sample extraction unit is used to perform acetonitrile double extraction and salting-out separation on apple samples. The purification unit is used to purify the concentrated and reconstituted extract with NH2 solid-phase extraction. The volume-degrading unit is used to dilute the purified sample to a final volume using n-heptane; The ultra-high performance phase chromatography detection unit includes a carbon dioxide supply module, a cosolvent supply module, a back pressure control module, a column temperature control module, an Acquity Trefoil CEL1 chiral chromatographic column, and a PDA detector, used to achieve the separation, detection, and quantitative analysis of four enantiomers of paclobutrazol and uniconazole in apples under the chromatographic conditions defined in claim 1.

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